U.S. patent application number 13/384981 was filed with the patent office on 2012-11-15 for therapeutic compositions and methods.
This patent application is currently assigned to BIOTHERA, INC.. Invention is credited to John P. Vasilakos.
Application Number | 20120288495 13/384981 |
Document ID | / |
Family ID | 43499417 |
Filed Date | 2012-11-15 |
United States Patent
Application |
20120288495 |
Kind Code |
A1 |
Vasilakos; John P. |
November 15, 2012 |
THERAPEUTIC COMPOSITIONS AND METHODS
Abstract
Methods relating to treating conditions that do not respond well
to EGF-r antagonist therapies are disclosed. Generally, the methods
include administering to a subject a composition that includes a
.beta.-glucan and, as either a second composition or a second
component of the composition, either antibody that binds to at
least one antigen specific to the KRAS-mutated cells and/or an
EGF-r antagonist.
Inventors: |
Vasilakos; John P.;
(Woodbury, MN) |
Assignee: |
BIOTHERA, INC.
Eagan
MN
|
Family ID: |
43499417 |
Appl. No.: |
13/384981 |
Filed: |
July 22, 2010 |
PCT Filed: |
July 22, 2010 |
PCT NO: |
PCT/US10/42925 |
371 Date: |
July 31, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61271514 |
Jul 22, 2009 |
|
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|
Current U.S.
Class: |
424/133.1 ;
424/138.1; 424/174.1; 514/54 |
Current CPC
Class: |
A61K 31/716 20130101;
A61P 35/00 20180101; A61K 39/39558 20130101; A61K 31/716 20130101;
A61P 35/04 20180101; A61K 39/39558 20130101; A61K 45/06 20130101;
A61K 2300/00 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
424/133.1 ;
424/174.1; 424/138.1; 514/54 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61P 35/00 20060101 A61P035/00; A61K 31/716 20060101
A61K031/716 |
Claims
1. A method comprising: administering to a subject having a tumor
comprising cells comprising a KRAS mutation: a composition
comprising a .beta.-glucan; and an antibody composition that binds
to at least one antigen specific to the KRAS-mutated cells; each in
an amount that, in combination with the other, is effective to
ameliorate at least one symptom or clinical sign associated with
the condition; with the proviso that the KRAS-mutated cells are not
SKOV-3 cells or NC1-H23 cells.
2. (canceled)
3. The method of claim 1 further comprising administering to the
subject a composition that includes a membrane-bound complement
regulatory protein (mCRP) antagonist.
4-5. (canceled)
6. The method of claim 1 wherein the condition comprises colon
carcinoma, colorectal carcinoma, lung adenocarcinoma, lung squamous
cell carcinoma, lung large cell carcinoma, intraductal papillary
mucinous tumor, or mucinous cystic tumor.
7. The method of claim 1 wherein the KRAS-mutated cells comprise at
least one of: GEO cells and HCT-115 cells.
8. The method of claim 1 wherein the antigen specific to
KRAS-mutated cells comprises epidermal growth factor receptor
(EGF-r).
9-11. (canceled)
12. The method of claim 8 wherein the antibody composition
comprises cetuximab, panitumumab, rituximab, bevacizumab,
trastuzumab, oralemtuzumab.
13. (canceled)
14. A method of improving treatment of condition that includes
administering an epidermal growth factor receptor (EGF-r)
antagonist to a subject, the method comprising: administering an
effective amount of a composition comprising a .beta.-glucan to the
subject, wherein the subject comprises neoplastic cells comprising
a KRAS mutation; with the proviso that the condition does not
comprise a tumor that comprises SKOV-3 cells or NC1-H23 cells.
15. (canceled)
16. The method of claim 14 further comprising administering to the
subject a composition that includes a mCRP antagonist.
17-18. (canceled)
19. The method of claim 14 wherein the condition comprises colon
carcinoma, colorectal carcinoma, lung adenocarcinoma, lung squamous
cell carcinoma, lung large cell carcinoma, intraductal papillary
mucinous tumor, or mucinous cystic tumor.
20. The method of claim 14 wherein the KRAS-mutated cells comprise
at least one of: GEO cells and HCT-115 cells.
21. The method of claim 14 wherein the EGF-r antagonist comprises
antibody that specifically binds EGF-r.
22-24. (canceled)
25. The method of claim 21 wherein the antibody comprises cetuximab
or panitumumab.
26-33. (canceled)
34. A method comprising: administering to a subject having a tumor
comprising cells comprising a KRAS mutation: a first composition
comprising a .beta.-glucan; and a second composition comprising an
epidermal growth factor receptor (EGF-r) antagonist; each in an
amount that, in combination with the other, is effective to
ameliorate at least one symptom or clinical sign associated with
the condition; with the proviso that the KRAS-mutated cells are not
SKOV-3 cells or NC1-H23 cells.
35. The method of claim 34 wherein the first composition and the
second composition are combined prior to being administered to the
subject.
36. The method of claim 34 further comprising administering to the
subject a composition that includes a mCRP antagonist.
37-38. (canceled)
39. The method of claim 34 wherein the condition comprises colon
carcinoma, colorectal carcinoma, lung adenocarcinoma, lung squamous
cell carcinoma, lung large cell carcinoma, intraductal papillary
mucinous tumor, or mucinous cystic tumor.
40. The method of claim 34 wherein the KRAS-mutated cells comprise
at least one of: GEO cells and HCT-115 cells.
41. The method of claim 34 wherein the EGF-r antagonist comprises
antibody.
42-44. (canceled)
45. The method of claim 41 wherein the antibody composition
comprises cetuximab or panitumumab.
46-48. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims priority to U.S. Provisional Patent
Application Ser. No. 61/271,514, filed Jul. 22, 2009.
BACKGROUND
[0002] Certain epidermal growth factor receptor (EGF-r) antagonists
are established therapeutic agents effective for treating certain
types of tumors. For example, cetuximab (ERBITUX, Bristol-Myers
Squibb, New York, N.Y.) and panitumumab (VECTIBIX, Amgen, Thousand
Oaks, Calif.) are EGF-r antagonist antibodies, each of which is
approved for the treatment of patients who have metastatic
colorectal cancer. However, certain patients with colorectal cancer
may not respond to treatment with panitumumab or cetuximab. These
patients often have tumors with KRAS mutations.
[0003] In metastatic colorectal cancer, EGF-r transmits signals
through a set of intracellular proteins, which instruct the cancer
cell to reproduce and metastasize. Blocking the EGF-r--such as by
using an EGF-r antagonist--can interfere with this malignant
signaling. However, in patients with mutant KRAS, the signaling
continues despite EGF-r antagonist therapy. Mutated KRAS genes have
been reported to be detected in about 40% of metastatic colorectal
cancer patients, depending on the testing used.
[0004] In addition, data from lung cancer trials to show that the
KRAS mutation is also associated with a lack of response to the
small-molecule compounds that inhibit EGF-r--e.g., erlotinib
(TARCEVA, Genentech, South San Francisco, Calif.) and gefitinib
(IRESSA, Astra Zeneca, Wilmington, Del.). Erlotinib is currently
approved for treatment of patients with locally advanced or
metastatic non-small cell lung cancer (NSCLC) or advanced,
unresectable, or metastatic pancreatic cancer. Gefitinib is
currently approved for the treatment of locally advanced NSCLC. The
KRAS-related data on erlotinib and gefitinib in treating NSCLC have
been reported to be consistent with the KRAS-related data on the
treatment of colorectal cancer with cetuximab or panitumumab for
these drugs to have any benefit, the tumors must possess wild-type
KRAS.
[0005] Consequently, a need exists for methods for treating
conditions associated with KRAS mutations--conditions that do not
respond well to EGF-r antagonist therapies.
SUMMARY OF THE INVENTION
[0006] In one aspect, the present invention provides methods
relating to treating a condition characterized, at least in part,
by the presence of cells that include at least one KRAS mutation.
Generally, the method can include administering to a subject having
a tumor comprising cells comprising a KRAS mutation one or more
compositions that, in total, include substances in amounts that, in
combination with the other, are effective to ameliorate at least
one symptom or clinical sign associated with the condition. One
composition includes a .beta.-glucan. In some embodiments, a second
composition includes an antibody composition that binds to at least
one antigen specific to the KRAS-mutated cells. In other
embodiments, a second composition can include an epidermal growth
factor receptor (EGF-r) antagonist. In either case, the composition
that includes a .beta.-glucan and the second composition can be
combined prior to being administered to the subject so that the
subject experiences a single administration of both
compositions.
[0007] In some embodiments, the method further includes
administering to the subject a composition that includes a
membrane-bound complement regulatory protein (mCRP) antagonist.
Prior to being administered to the subject, the composition that
includes a mCRP antagonist may be combined with the composition
that includes the .beta.-glucan, the second composition, or
both.
[0008] In some embodiments, the condition can include colon
carcinoma, colorectal carcinoma, lung, adenocarcinoma, lung
squamous cell carcinoma, lung large cell carcinoma, intraductal
papillary mucinous tumor, and/or mucinous cystic tumor.
[0009] In embodiments in which the second composition includes
antibody, the antibody can include a monoclonal antibody, a
humanized monoclonal antibody, a recombinant antibody, or any
combination of two or more types of antibody. In certain
embodiments, the antibody can include cetuximab.
[0010] In some embodiments, the method can further include
identifying the subject as having the condition that includes a
tumor having cells that include a KRAS mutation.
[0011] In another aspect, the invention provides a method that
generally includes providing to a subject having the condition a
composition that comprises a .beta.-glucan, wherein administering
to the subject an amount of the composition that, in combination
with an antibody composition that specifically binds to at least
one antigen specific to the KRAS-mutated cells, is effective to
ameliorate at least one symptom or clinical sign of the condition.
In some embodiments, a second composition includes an antibody
composition that binds to at least one antigen specific to the
KRAS-mutated cells. In other embodiments, a second composition can
include an epidermal growth factor receptor (EGF-r) antagonist. In
either case, the composition that includes a .beta.-glucan and the
second composition can be combined prior to being administered to
the subject so that the subject experiences a single administration
of both compositions.
[0012] In another aspect, the invention provides a method of
improving treatment of condition that includes administering an
epidermal growth factor receptor (EGF-r) antagonist to a subject.
Generally, the method includes administering an effective amount of
a composition that includes a .beta.-glucan to the subject, wherein
the subject comprises neoplastic cells that include a KRAS
mutation.
[0013] In some embodiments, the composition comprising the
.beta.-glucan may be combined with the EGF-r antagonist prior to
being administered to the subject.
[0014] In some embodiments, the method further includes
administering to the subject a composition that includes a mCRP
antagonist. The mCRP may be combined with the .beta.-glucan, the
EGF-r antagonist, or both, prior to being administered to the
subject.
[0015] In some embodiments, the condition can include colon
carcinoma, colorectal carcinoma, lung adenocarcinoma, lung squamous
cell carcinoma, lung large cell carcinoma, intraductal papillary
mucinous tumor, and/or mucinous cystic tumor.
[0016] In some embodiments, the EGF-r antagonist can include
antibody that specifically binds EGF-r. When the EGF-r antagonist
includes antibody, the antibody can include a monoclonal antibody,
a humanized monoclonal antibody, a recombinant antibody, or any
combination of two or more types of antibody. In certain
embodiments, the antibody can include cetuximab.
[0017] The above summary of the present invention is not intended
to describe each disclosed embodiment or every implementation of
the present invention. The description that follows more
particularly exemplifies illustrative embodiments. In several
places throughout the application, guidance is provided through
lists of examples, which examples can be used in various
combinations. In each instance, the recited list serves only as a
representative group and should not be interpreted as an exclusive
list.
BRIEF DESCRIPTION OF THE FIGURES
[0018] FIG. 1 is a line graph showing the results of an in vivo
xenograft model in which tumor cells harboring a KRAS mutation were
grafted subcutaneously into mice and then subsequently treated with
an EGF-r antagonist antibody, a .beta.-glucan, a combination of the
EGF-r antibody and .beta.-glucan, or saline. Therapy was initiated
after the tumor reached 350 mm.sup.3. Tumor: GEO colon carcinoma
KRAS mutant; Mice: Nu/Nu; Monoclonal antibody: ERBITUX, 0.25
mg/dose; Regimen: intravenous administration, 2.times./wk, 4 wks
(q3d.times.8 doses).
[0019] FIG. 2 shows flow cytometry data demonstrating that a
.beta.-glucan composition enhances the ability of human peripheral
blood mononuclear cells (PBMCs) to kill KRAS-mutated tumor cells in
vitro.
[0020] FIG. 3 shows data demonstrating that a .beta.-glucan
composition enhances the ability of human peripheral blood
mononuclear cells (PBMCs) to kill KRAS-mutated tumor cells in
vitro. (N=1; E:T=25:1).
[0021] FIG. 4 shows data demonstrating that a .beta.-glucan
composition exhibits greater antitumor activity in the presence of
an EGF-r antagonist antibody. (N=1; E:T=25:1).
DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0022] The present invention relates to methods that can address
the need to provide therapies for treating conditions that do not
respond well to EGF-r antagonist therapies. Such conditions can
include certain cancers such as, for example, colorectal cancer,
pancreatic cancer, and non-small cell lung cancer. Generally, the
methods include administering to a subject a composition that
includes a .beta.-glucan and, as either a second composition or a
second component of the composition, antibody that binds to at
least one antigen specific to the KRAS-mutated cells and/or an
EGF-r antagonist. Administering additional components and/or
compositions is also possible.
[0023] The term "and/or" means one or all of the listed elements or
a combination of any two or more of the listed elements.
[0024] The terms "comprises" and variations thereof do not have a
limiting meaning where these terms appear in the description and
claims.
[0025] Unless otherwise specified, "a," "an," "the," and "at least
one" are used interchangeably and mean one or more than one.
[0026] Also herein, the recitations of numerical ranges by
endpoints include all numbers subsumed within that range (e.g., 1
to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).
[0027] For any method disclosed herein that includes discrete
steps, the steps may be conducted in any feasible order. And, as
appropriate, any combination of two or more steps may be conducted
simultaneously.
[0028] In a first aspect, the invention provides a method that can
ameliorate at least one symptom or clinical sign associated with a
condition that is at least partially characterized by the presence
of tumor cells that include a KRAS mutation. As used herein, cells
that possess a KRAS mutation (or a similar phrase such as
"KRAS-mutated cells") can include cells that include a mutation in
the KRAS gene. The KRAS gene is one of a family (i.e., the RAS
family) of genes that encode a family of membrane-associated
GTPases, proteins that regulate signal transduction upon binding of
ligand to a variety of membrane receptors. In the scientific
literature, KRAS is also written as K-RAS, Ki-RAS, and V-H-Ras.
Generally, the method includes administering to a subject having
such a condition one or more compositions. One composition includes
a .beta.-glucan. Another composition includes antibody that binds
to at least one antigen specific to the KRAS-mutated cells. Each of
the .beta.-glucan and antibody is provided in an amount that, in
combination with the other, is effective to ameliorate at least one
symptom or clinical sign associated with the condition.
[0029] As used herein, the term "ameliorate" can refer to any
reduction in the extent, severity, frequency, and/or likelihood of
a symptom or clinical sign characteristic of a particular
condition. The term "symptom" can refer to any subjective evidence
of disease or of a patient's condition--e.g., pain; while the terms
"sign" and/or "clinical sign" can refer to an objective physical
finding relating to a particular condition capable of being found
by one other than the patient--e.g., tumor diameter, tumor volume,
and the like.
[0030] As used herein, the term "antibody" can refer to a
monoclonal antibody, a mixture of a plurality of monoclonal
antibodies, one or more recombinantly-produced antibodies, one or
more synthetically produced antibodies, one or more antibodies of a
polyclonal composition, or any combination of two or more of the
foregoing. Antibody can be specific for a particular target such
as, for example, an antigen. As used herein, the term "specific"
when used to characterize the affinity of antibody for a
target--e.g., immunospecific--can refer to the antibody possessing
a differential (e.g., preferential) or a non-general affinity for
the target. For example, antibody "specific" to a particular target
is antibody that binds to the target with greater affinity, to any
degree, than background affinity for the target. As used herein,
the term "antigen" can refer to any substance that may be bound by
antibody in a manner that is immunospecific to some degree. Also,
the term "specific" when used to characterize expression of an
antigen by a particular cell type is an antigen that is
differentially expressed by the particular cell type to any
exploitable degree. Thus, an antibody composition that specifically
binds to at least one antigen specific to KRAS-mutated cells refers
to antibody that differentially binds to a target antigen (e.g.,
the antibody binds to the target antigen with an affinity that is
greater than background) that is specific to KRAS-mutated cells
(e.g., the antigen is expressed by KRAS-mutated cells to an
exploitably greater degree than the antigen is expressed by normal
cells).
[0031] In some embodiments, the KRAS-mutated cells are cells other
than SKOV-3 cells or NC1-H23 cells.
[0032] The .beta.-glucan and compositions that include
.beta.-glucan, are described in detail below.
[0033] The antibody composition can include either a single
antibody or a plurality of antibodies. The antibody composition can
include a monoclonal antibody such as, for example, a humanized
monoclonal antibody. In some embodiments, the antibody can include
a recombinant antibody. In some embodiments, the antibody can
include a therapeutic antibody such as, for example, cetuximab,
panitumumab, rituximab, bevacizumab, trastuzumab, alemtuzumab, and
the like, that have received regulatory approval for use in
treating one or more conditions such as, for example, certain
cancers. In some embodiments, the antibody can include one or more
polyclonal antibodies. Standard methods for producing monoclonal
antibodies--including humanize monoclonal antibodies, recombinant
antibodies, and polyclonal antibodies against a particular target
are known in the art. The antibody composition can include any
combination of two or more antibodies.
[0034] In some embodiments, the .beta.-glucan composition and the
antibody composition may be administered separately. In other
embodiments, the .beta.-glucan composition and the second
composition can be combined prior to being administered to the
subject. In this way, the subject can receive the benefit of both
compositions but experience only a single administration. Exemplary
methods of administering the .beta.-glucan composition and the
antibody composition are described in more detail below.
[0035] In some embodiments, the method further includes
administering to the subject a composition that includes a
membrane-bound complement regulatory protein (mCRP) antagonist. A
"mCRP antagonist" can refer to a compound that can combine with a
mCRP to inhibit biological and/or biochemical activity of the mCRP.
A mCRP antagonist may be a ligand that directly binds to the mCRP.
"Inhibit" and variations thereof can refer to any measurable
reduction of biological and/or biochemical activity. For example,
inhibition of a mCRP can refer to a decrease in the extent to which
mCRP inhibits complement activation--i.e., mCRP inhibition results
in increased complement activation. The extent of inhibition may be
characterized as a percentage of a normal level of activity.
[0036] The mCRP antagonist can be provided in a composition
separate from the .beta.-glucan composition and/or the antibody
composition. Alternatively, the mCRP antagonist, when present, can
be combined with the .beta.-glucan composition, the antibody
composition, or both.
[0037] In some embodiments, the condition can include certain forms
of cancer such as, for example, colon carcinoma, colorectal
carcinoma, lung cancer (e.g., lung adenocarcinoma, lung squamous
cell carcinoma and/or lung large cell carcinoma), breast cancer
(e.g., intraductal papillary mucinous tumor and/or mucinous cystic
tumor), oral cancer, ovarian cancer, prostate cancer, pancreatic
cancer, multiple myeloma, malignant melanoma and/or non-melanoma
skin cancers.
[0038] In some embodiments, the KRAS-mutated cells include GEO
cells and/or HCT-115 cells.
[0039] In some embodiments, the antibody can bind to a
KRAS-specific antigen such as, for example, EGF-r. Antibodies that
bind to EGF-r, including therapeutic antibodies that have received
regulatory approval for the treatment of certain conditions, are
described in more detail below.
[0040] Despite the effectiveness of antibodies that bind to EGF-r
for treating some cancer patients, certain patients may not respond
to treatment with, for example, panitumumab or cetuximab. These
patients often have tumors that harbor KRAS mutations. Thus, in
some embodiments, the method can include improving the efficacy of
anti-EGF-r antibody therapy--i.e., improving treatment of condition
that includes administering an anti-EGF-r antibody to a patient
where the anti-EGF-r antibody, alone, in insufficiently effective.
Generally, the method includes administering an effective amount of
a composition that includes a .beta.-glucan to a patient that has a
condition characterized, at least in part, by neoplastic cells that
harbor a KRAS mutation and for whom anti-EGF-r antibody therapy has
been unsuccessful.
[0041] Thus, in some embodiments, the method can further include
identifying a patient as having a condition that is characterized,
at least in part, by the presence of cells that harbor a KRAS
mutation. Standard methods for detecting a KRAS mutation in a cell
are known in the art. For example, KRAS mutations may be identified
from cancer tissue or cells obtained from patients using a
DNA-based assay that detects mutations in the KRAS gene. Such
assays may be PCR-based such as, for example, the TheraScreen: KRAS
Mutation Test Kit from DxS Ltd, Manchester, UK. The TheraScreen:
KRAS Mutation Test Kit is approved by the FDA for testing clinical
samples for KRAS mutations.
[0042] In another aspect, the invention provides a method that can
ameliorate at least one symptom or clinical sign associated with a
condition that is at least partially characterized by the presence
of tumor cells that include a KRAS mutation. Generally, the method
includes administering to a subject having such a condition one or
more compositions. One composition includes a .beta.-glucan.
Another composition includes an EGF-r antagonist. Each of the
.beta.-glucan and the EGF-r antagonist is provided in an amount
that, in combination with the other, is effective to ameliorate at
least one symptom or clinical sign associated with the
condition.
[0043] As used herein, an "EGF-r antagonist" can include a compound
that can combine with EGF-r to inhibit cellular activity (e.g.,
cell signaling) that would otherwise result from an agonist-EGFr
interaction. The extent of inhibition may be characterized as a
percentage of a normal level of activity.
[0044] EGF-r antagonists include antibodies that bind to EGF-r
(e.g., cetuximab and panitumumab) as well as small molecules
antagonists such as, for example, erlotinib and gefitinib.
[0045] In some embodiments, the KRAS-mutated cells are cells other
than SKOV-3 cells or NC1-H23 cells.
[0046] The .beta.-glucan, compositions that include .beta.-glucan,
and exemplary EGF-r antagonists are described in detail below. In
some embodiments, the EGF-r antagonist may include antibody that
binds to EGF-r. An EGF-r antagonist antibody can include either a
single antibody or a plurality of antibodies. An EGF-r antagonist
antibody can include a monoclonal antibody such as, for example, a
humanized monoclonal antibody. In some embodiments, the EGF-r
antagonist antibody can include a recombinant antibody. In some
embodiments, the EGF-r antagonist antibody can include a
therapeutic antibody such as, for example, cetuximab, panitumumab,
rituximab, bevacizumab, trastuzumab, alemtuzumab, and the like,
that have received regulatory approval for use in treating one or
more conditions such as, for example, certain cancers. In some
embodiments, the EGF-r antagonist antibody can include one or more
polyclonal antibodies. Standard methods for producing monoclonal
antibodies--including humanize monoclonal antibodies, recombinant
antibodies, and polyclonal antibodies against a particular target
are known in the art. The EGF-r antagonist composition can include
any combination of two or more antibodies.
[0047] In some embodiments, the .beta.-glucan composition and the
EGF-r antagonist composition may be administered separately. In
other embodiments, the .beta.-glucan composition and the EGF-r
antagonist composition can be combined prior to being administered
to the subject. In this way, the subject can receive the benefit of
both compositions but experience only a single administration.
Exemplary methods of administering the .beta.-glucan composition
and the EGF-r composition are described in more detail below.
[0048] In some embodiments, the method further includes
administering to the subject a composition that includes a mCRP
antagonist. The mCRP antagonist can be provided in a composition
separate from the .beta.-glucan and/or the EGF-r antagonist.
Alternatively, the mCRP antagonist, when present, can be combined
with the .beta.-glucan composition, the EGF-r antagonist
composition, or both.
[0049] In some embodiments, the condition can include certain forms
of cancer such as, for example, colon carcinoma, colorectal
carcinoma, lung cancer (e.g., lung adenocarcinoma, lung squamous
cell carcinoma and/or lung large cell carcinoma), breast cancer
(e.g., intraductal papillary mucinous tumor and/or mucinous cystic
tumor), oral cancer, ovarian cancer, prostate cancer, pancreatic
cancer, multiple myeloma, malignant melanoma and/or non-melanoma
skin cancers. In some embodiments, the KRAS-mutated cells include
GEO cells and/or HCT-115 cells.
[0050] Despite the effectiveness of EGF-r antagonists for treating
some cancer patients, certain patients may not respond to treatment
with, for example, panitumumab or cetuximab. These patients often
have tumors that harbor KRAS mutations. Thus, in some embodiments,
the method can include improving the efficacy of EGF-r antagonist
therapy--i.e., improving treatment of condition that includes
administering an EGF-r antagonist to a patient where the EGF-r
antagonist, alone, in insufficiently effective. Generally, the
method includes administering an effective amount of a composition
that includes a .beta.-glucan to a patient that has a condition
characterized, at least in part, by neoplastic cells that harbor a
KRAS mutation and for whom EGF-r antagonist therapy has been
unsuccessful.
[0051] In another aspect, the invention provides a method the
includes, generally, providing a composition that includes a
.beta.-glucan to a subject having a condition that is
characterized, at least in part, by a tumor that includes
KRAS-mutated cells, wherein administering to the subject an amount
of the composition that, in combination with a second composition,
is effective to ameliorate at least one symptom or clinical sign of
the condition, wherein the second composition includes an EGF-r
antagonist and/or antibody that binds to at least one antigen
specific to the KRAS-mutated cells.
[0052] In some embodiments, the .beta.-glucan composition further
includes a mCRP antagonist.
[0053] The second composition can include any one or more of the
EGF-r antagonists described herein, any one or more of the antibody
compositions that include antibody that binds to at least one
antigen specific to the KRAS-mutated cells described herein, or any
combination thereof. In some embodiments, the second composition
can further include a mCRP antagonist.
[0054] In some embodiments, the .beta.-glucan composition and the
second composition may be provided separately. In other
embodiments, the .beta.-glucan composition and the second
composition can be combined prior to being provided to the
subject.
[0055] In some embodiments, the second composition can include
antibody that binds to EGF-r such as, for example, a therapeutic
antibody such as, for example, cetuximab or panitumumab that have
received regulatory approval for use in treating one or more
conditions such as, for example, certain cancers.
[0056] In some embodiments, the second composition can include
antibody that binds to at least one antigen specific to
KRAS-mutated cells such as, for example, cetuximab, panitumumab,
rituximab, bevacizumab, trastuzumab, alemtuzumab, and the like.
[0057] In some embodiments, the KRAS-mutated cells are cells other
than SKOV-3 cells or NC1-H23 cells.
[0058] In each aspect of the invention, the various compositions
may, if desired, be provided in a pack or dispenser device and/or a
kit which may contain one or more unit dosage forms containing the
active ingredients. The pack may, for example, include metal or
plastic foil such as, for example, a blister pack. The pack or
dispenser device may be accompanied by instructions for
administration.
[0059] A composition that includes .beta.-glucan can include PGG
(poly-(1-6)-.beta.-D-glucopyranosyl-(1-3)-.beta.-D-glucopyranose),
neutral soluble .beta.-glucan, triple helical .beta.-glucan
(BETAFECTIN, Biopolymer Engineering, Inc., Eagan, Minn.),
.beta.-glucans of various aggregate numbers, and any combination
thereof.
[0060] The .beta.-glucan preparations may be prepared from
insoluble glucan particles. A .beta.-glucan polysaccharide can
exist in one of at least four distinct conformations: single
disordered chains, single helix, single triple helix, and triple
helix aggregates. As used herein, the term "single triple helix"
refers to a .beta.-glucan conformation in which three single chains
are joined together to form a triple helix structure. In a single
triple helix conformation, there is no higher ordering of the
triple helices--i.e., there is substantially no aggregation of
triple helices. As used herein, the term "triple helix aggregate"
refers to a .beta.-glucan conformation in which two or more triple
helices are joined together via non-covalent interactions. A
.beta.-glucan composition can include one or more of these forms,
depending upon such conditions as pH and temperature.
[0061] As used herein, the "molecular weight" of a .beta.-glucan
composition is the mass average molar mass of the collection of
polymer molecules within the composition. The characterization of a
collection of polymer molecules in terms of polymer mass average
molar mass is well known in the art of polymer science. The terms
"neutral soluble .beta.-glucan" and "neutral soluble glucan" refer
to an aqueous soluble .beta.-glucan having a unique triple helical
conformation that results from the denaturation and re-annealing of
aqueous soluble glucan.
[0062] The .beta.-glucan may be formed from starting material that
includes glucan particles such as, for example, whole glucan
particles described by U.S. Pat. No. 4,810,646; U.S. Pat. No.
4,992,540; U.S. Pat. No. 5,082,936; and/or U.S. Pat. No. 5,028,703.
The source of the whole glucan particles can be the broad spectrum
of glucan-containing fungal organisms that contain .beta.-glucans
in their cell walls. Suitable sources for the whole glucan
particles include, for example, Saccharomyces cerevisiae R4
(deposit made in connection with U.S. Pat. No. 4,810,646;
Agricultural Research Service No. NRRL Y-15903) and R4Ad (American
Type Culture Collection, Manassas, Va., ATCC No. 74181). The
structurally modified glucans hereinafter referred to as "modified
glucans" derived from S. cerevisiae R4 can be potent immune system
activators (U.S. Pat. No. 5,504,079).
[0063] The whole glucan particles from which the .beta.-glucan may
be prepared can be in the form of a dried powder, as described in
U.S. Pat. No. 4,810,646; U.S. Pat. No. 4,992,540; U.S. Pat. No.
5,082,936; and/or U.S. Pat. No. 5,028,703. In order to prepare the
.beta.-glucan, however, it is not necessary to perform the final
organic extraction and wash steps described in one or more of these
patent documents.
[0064] In some embodiments, soluble glucans can include branched
polymers of glucose, referred to as PGG, containing .beta.(1-3) and
.beta.(1-6) linkages in varying ratios depending on the organism
from which they are obtained and the processing conditions
employed. PGG glucan preparations can contain neutral glucans,
which have not been modified by substitution with functional (e.g.,
charged) groups or other covalent attachments. The biological
activity of PGG glucan can be controlled by varying the average
molecular weight and the ratio of .beta.(1-6) to .beta.(1-3)
linkages of the glucan molecules. (U.S. Pat. No. 4,810,646; U.S.
Pat. No. 4,992,540; U.S. Pat. No. 5,082,936; and U.S. Pat. No.
5,028,703). The average molecular weight of soluble glucans
generally can be from about 10,000 daltons to about 500,000
daltons. In some embodiments, the average molecular weight of
soluble glucans can be from about 30,000 daltons to about 50,000
daltons.
[0065] Soluble .beta.-glucan (e.g., IMPRIME PGG, Biopolymer
Engineering, Inc., Eagan, Minn.) has been shown to increase the
number of neutrophils and monocytes as well as their direct
antitumor activity (e.g., phagocytosis and microbial killing).
However, the soluble .beta.-glucan does not stimulate the
production of biochemical mediators, such as, for example, IL-1,
TNF, and leukotrienes. When produced, these biological mediators
can cause side effects such as high fever, inflammation, wasting
disease, and organ failure. Also, soluble .beta.-glucan lacks the
toxicity common to many immunomodulators.
[0066] Soluble .beta.-glucans can be composed of glucose monomers
organized as a .beta.(1-3) linked glucopyranose backbone with
periodic branching via .beta.(1-6) glycosidic linkages. The soluble
glucan preparations contain glucans that have not been
substantially substituted with functional (e.g., charged) groups or
other covalent attachments. One biologically active form of soluble
.beta.-glucan is produced by dissociating the native glucan
conformations and then re-annealing and purifying the resulting
triple helical conformation. The triple helical conformation of the
soluble glucan contributes to the glucan's ability to selectively
activate the immune system without stimulating the production of
detrimental biochemical mediators. Methods of making soluble
.beta.-glucans are described in Example 1.
[0067] Soluble glucan preparations can be prepared from insoluble
glucan particles such as, for example, insoluble glucan particles
derived from yeast organisms as described herein. Other strains of
yeast from which insoluble glucan particles can be obtained
include, for example, Saccharomyces delbrueckii, Saccharomyces
rosei, Saccharomyces microellipsodes, Saccharomyces carlsbergensis,
Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces
fragilis, Kluyveromyces polysporus, Candida albicans, Candida
cloacae, Candida tropicalis, Candida utilis, Hansenula wingeri,
Hansenula arni, Hansenula henricii, Hansenula americana.
[0068] As described above, certain conformation of .beta.-glucan
can include aggregates of single chain such as, for example, a
single triple helix (an aggregate of three single helices) or a
triple helix aggregate (an aggregate of triple helices). The
"aggregate number" of a .beta.-glucan conformation is the number of
single chains which are joined together in that conformation. For
example, the aggregate number of a single helix is 1, the aggregate
number of a single triple helix is 3, and the aggregate number of a
triple helix aggregate is greater than 3. For example, a triple
helix aggregate consisting of two triple helices joined together
has an aggregate number of 6.
[0069] The aggregate number of a .beta.-glucan sample under a
specified set of conditions can be determined by determining the
average molecular weight of the polymer under those conditions. The
.beta.-glucan is then denatured, that is, subjected to conditions
which separate any aggregates into their component single polymer
chains. The average molecular weight of the denatured polymer is
then determined. The ratio of the molecular weights of the
aggregated and denatured forms of the polymer is the aggregate
number. A typical .beta.-glucan composition includes molecules
having a range of chain lengths, conformations and molecular
weights. Thus, the measured aggregate number of a .beta.-glucan
composition is the mass average aggregate number across the entire
range of .beta.-glucan molecules within the composition. It is to
be understood that any reference herein to the aggregate number of
a .beta.-glucan composition refers to the mass average aggregate
number of the composition under the specified conditions. The
aggregate number of a composition indicates which conformation is
predominant within the composition. For example, a measured
aggregate number of about 6 or more is characteristic of a
composition in which the .beta.-glucan is substantially in the
triple helix aggregate conformation. Methods for preparing
.beta.-glucan aggregates are described in Example 2.
[0070] The conformation of a PGG-glucan preparation can be
temperature dependent; PGG-glucan can be predominantly in a triple
helix aggregate conformation at 25.degree. C., but can be a mixture
of triple helix aggregates and the single triple helix conformation
37.degree. C. (U.S. Patent Application Publication No. 2007/0059310
A1).
[0071] In certain embodiments, the soluble .beta.-glucan can be
substantially in a triple helix aggregate conformation under
physiological conditions--e.g., physiological pH, about pH 7, and
physiological temperature, about 37.degree. C. In one particular
embodiment, the .beta.-glucan consists essentially of .beta.-glucan
chains in one or more triple helix aggregate conformations under
physiological conditions.
[0072] As used herein, a soluble .beta.-glucan composition is
"substantially in a triple helix conformation" if greater that
about 50% by weight of the composition is in a triple helix
aggregate conformation under physiological conditions. In some
embodiments, the amount of .beta.-glucan in a triple helix
aggregate conformation under physiological conditions can be
greater than about 60% by weight such as, for example, greater than
about 70% by weight. In one embodiment, the soluble .beta.-glucan
composition can be characterized by an aggregate number under
physiological conditions of greater than about 6 such as, for
example, an aggregate number of the .beta.-glucan composition under
physiological conditions of at least about 7, at least about 8, or
at least about 9.
[0073] The methods described herein enhance the immune response in
humans and animals. Treatment of a condition characterized, at
least in part, by the presence of a tumor that includes
KRAS-mutated cells can involve activating the patient's immune
system against the KRAS-mutated cells. As used herein, "treat,"
"treating," and variations thereof can refer to reducing, limiting
progression, ameliorating, and/or resolving, to any extent, the
symptoms or clinical signs related to a condition. As used herein,
"treatment" can refer to any method for treating a condition. In
certain contexts, "treatment" may refer to any single step of such
a method such as, for example, administering a dose of a
composition.
[0074] The .beta.-glucan can be formulated for administering the
.beta.-glucan to a subject. The formulation can include one or more
suitable carriers or excipients.
[0075] The .beta.-glucan composition generally can be administered
to an animal or a human in an amount that, in combination with the
second composition, is effective to ameliorate at least one symptom
or clinical sign of the condition. The mode of administration of
the .beta.-glucan can be oral, enteral, parenteral, intravenous,
subcutaneous, intraperitoneal, intramuscular, topical, or
intranasal. The form in which the .beta.-glucan can be administered
(e.g., powder, tablet, capsule, solution, emulsion) can be
influenced by the route by which it is administered. The quantity
of .beta.-glucan to be administered can be determined on an
individual basis, and can be at least in part influenced by the
severity of the condition, the patient's condition and/or overall
health, the patient's weight, and the identity and efficacy of
other components of the treatment (e.g., EGF-r antagonist
composition, antibody composition, mCRP antagonist, etc.). In
general, a single dose can contain approximately 0.01 mg to
approximately 10 mg of modified glucan per kilogram of body weight
(mg/kg), although the methods described herein can be practiced
using doses of .beta.-glucan outside of this range. In some
embodiments, a single dose of .beta.-glucan can include from about
0.1 mg/kg to about 2.5 mg/kg such as, for example, from about 0.25
mg/kg to about 2 mg/kg.
[0076] The dosage for topical application can be from about 0.001
mg to about 2 mg per milliliter of carrier (mg/ml), although the
methods described herein can be practiced using a concentration of
.beta.-glucan outside of this range. In some embodiments, the
methods include a topical dose of, for example, from about 0.01
mg/ml to about 0.5 mg/ml.
[0077] In some embodiments, the .beta.-glucan composition generally
can be administered parenterally by injection such as, for example,
subcutaneously, intravenously, intramuscularly, intraperitoneally,
topically, orally or intranasaly. The .beta.-glucan composition can
be administered as a solution having a .beta.-glucan concentration
of from about 1 mg/ml to about 100 mg/ml such as, for example, a
.beta.-glucan concentration of 1 mg/ml to about 20 mg/ml or, for
example, a .beta.-glucan concentration of 1 mg/ml to about 5 mg/ml.
The solvent can be a physiologically acceptable aqueous medium such
as, for example, water, saline, PBS, or a 5% dextrose solution.
[0078] In general, the composition of the present invention can be
administered to an individual periodically as necessary to
ameliorate at least one symptom or clinical sign of the condition.
An individual skilled in the medical arts will be able to determine
the length of time during which the composition--or, in embodiments
in which more than one composition is administered, each
composition--is administered and the dosage, depending on the
physical condition of the patient and the disease or disorder being
treated.
[0079] The .beta.-glucan composition can optionally include other
components. The other components that can be included in a
particular composition can be influenced by the manner in which the
composition is to be administered. For example, a composition to be
administered orally in tablet form can include, in addition to
neutral soluble .beta.-glucan, a filler (e.g., lactose), a binder
(e.g., carboxymethyl cellulose, gum arabic, gelatin), an adjuvant,
a flavoring agent, a coloring agent and a coating material (e.g.,
wax or plasticizer). A .beta.-glucan composition to be administered
in liquid form can include neutral soluble .beta.-glucan and,
optionally, an emulsifying agent, a flavoring agent, and/or a
coloring agent. A .beta.-glucan composition for parenteral
administration can be mixed, dissolved, or emulsified in water,
sterile saline, phosphate buffered saline (PBS), dextrose or other
biologically acceptable carrier. A composition for topical
administration can be formulated into a gel, ointment, lotion,
cream, or other form in which the composition is capable of coating
the site to be treated.
[0080] The second composition, whether an EGF-r antagonist or
antibody that binds to an antigen that is specific to KRAS-mutated
cells, can be formulated for administering the second composition
to a subject. The formulation can include one or more suitable
carriers or excipients such as, for example, those just described
in connection with the .beta.-glucan composition.
[0081] In some embodiments, the second composition can include
antibody that binds to an antigen specific to KRAS-mutated cells.
As indicated above, the term "specific" when used to characterize
expression of an antigen by a particular cell type is an antigen
that is differentially expressed by the particular cell type to any
exploitable degree. One such antigen is EGF-r, which is expressed
to a greater degree in tumor cells--including KRAS-mutated tumor
cells--than normal (i.e., nontumor) cells.
[0082] In some embodiments, the second composition can include one
or more EGF-r antagonists. Suitable EGF-r antagonists used in the
composition of the invention include polyclonal and monoclonal
antibodies, recombinant human/mouse chimeric monoclonal antibody
(e.g., cetuximab), antibody fragments, other proteins, and small
molecules that bind specifically to the extracellular domain of the
human epideimal growth factor receptor. The EGF-r antagonists can
be administered separately or in various combinations.
[0083] Suitable EGF-r antagonists include anti-EGF-r antibodies
such as, for example, cetuximab (ERBITUX, Bristol-Myers Squibb, New
York, N.Y.) and panitumumab (VECTIBIX, Amgen, Thousand Oaks,
Calif.). Cetuximab is a recombinant, mouse/human chimeric
monoclonal antibody which binds to the extracellular domain of the
human EGF-r, blocking the binding of EGF to its receptor, thereby
inhibiting growth in cells which express EGF-r, such as tumor
cells. U.S. Pat. No. 6,217,866 discloses two monoclonal antibodies
numbered 96 and 108 from cell lines ATCC HB 9763 and 9764,
respectively. Both antibodies are contemplated as EGF-r antagonists
suitable for use in the methods described herein. Panitumumab is a
fully human monoclonal antibody against human EGF-r described in
U.S. Pat. No. 6,235,883.
[0084] Suitable EGF-r antagonists also include other monoclonal
antibodies, polyclonal antibodies, antibody fragments, or other
proteins or small molecules. Suitable EGF-r antagonists include
compounds that can combine with the EGF-r to inhibit cellular
activity (e.g., cell signaling) that would otherwise result from an
agonist-receptor interaction.
[0085] In some embodiments, the EGF-r antagonist can be monoclonal
or polyclonal antibody that bind to the EGF-r of various species
including, for example, rat, mouse, horse, cow, goat, sheep, pig
and/or rabbit. In other embodiments, the EGF-r antagonist can be a
chimeric antibody (e.g., cetuximab) produced from a human antibody
and a monoclonal antibody made in any of the animals identified
above. In other embodiments, an EGF-r antagonist can include an
antibody fragment such as, for example, a variable region from an
antibody that binds to EGF-r. In other embodiments, an EGF-r
antagonist can include an epidermal growth factor (EGF) mutant
that, while still able to bind to EGF-r, the binding of the mutant
EGF to EGF-r does not result in EGF-r-mediated cell signaling and
blocks binding of wild-type EGF to EGF-r. In some embodiments, the
EGF-r antagonist can be a small molecule that binds to EGF-r and
blocks EGF signaling through the EGF-r such as, for example,
erlotinib (TARCEVA, Genentech, South San Francisco, Calif.) and
gefitinib (IRESSA, Astra Zeneca, Wilmington, Del.). In still other
embodiments, the EGF-r antagonist can include a soluble EGF-r
fragment such as, for example, a fragment encompassing the
extracellular domain of the EGF-r, which can bind EGF thereby
limiting the amount of free EGF available to bind to EGF-r.
[0086] Certain EGF-r antagonists have been granted regulatory
approval for treating cancer. For example, cetuximab has received
regulatory approval for treating colon cancer. Other cancers that
can be treated by EGF-r antagonists include, for example, ovarian,
breast, prostate, colon, pancreatic, oral epidermoid, multiple
myeloma, malignant melanoma, and non-melanoma skin cancers. As
described in the Background section, EGF-r antagonists, alone, can
be ineffective for treating a condition characterized, at least in
part, by a tumor that includes KRAS-mutated cells. Indications
treatable by practicing the methods described herein include forms
of the identified cancers that are characterized, at least in part,
by tumors that include cells harboring at least one KRAS
mutation.
[0087] The EGF-r antagonist may be administered as directed for the
particular EGF-r antagonist. For example, the recommended dose of
cetuximab is 400 mg/m.sup.2 as an initial loading dose (first
infusion) administered as a 120-minute intravenous (IV) infusion
(maximum infusion rate 5 ml/min). The recommended weekly
maintenance dose (all other infusions) is 250 mg/m.sup.2 infused
over 60 minutes (maximum infusion rate 5 ml/min). Following a
two-hour infusion of 400 mg/m.sup.2 of cetuximab, the maximum mean
serum concentration (Cmax) was 184 .mu.g/ml (range: 92-327
.mu.g/ml) and the mean elimination half-life was 97 hours (range
41-213 hours). A one-hour infusion of 250 mg/m.sup.2 produced a
mean Cmax of 140 .mu.g/mL (range 120-170 .mu.g/ml). Following the
recommended dose regimen (400 mg/m.sup.2 initial dose/250
mg/m.sup.2 weekly dose), cetuximab concentrations reached
steady-state levels by the third weekly infusion with mean peak and
trough concentrations across studies ranging from 168 to 235 and 41
to 85 .mu.g/ml, respectively. The mean half-life was 114 hours
(range 75-188 hours). Other administration methods are contemplated
as described below.
[0088] The methods described herein involve administering to a
subject a combination of a .beta.-glucan composition and a second
composition that includes either an EGF-r antagonist or antibody
that binds to an antigen specific to KRAS-mutated cells. Any of the
above described .beta.-glucans can be combined with any of the
above described EGF-r antagonists or any antibody that binds to any
antigen specific to KRAS-mutated cells. As discussed above, the
compositions can be combined prior to being administered to a
subject. The combination of the .beta.-glucan and the second
composition can ameliorate at least one symptom or clinical sign of
the condition, particularly when an EGF-r antagonist, if
administered alone or in combination with some other active agent,
is insufficient to do so. In some embodiments, a subject is first
identified (e.g., diagnosed) as having a condition that is
characterized, at least in part, by a tumor having cells that
harbor at least one KRAS mutation.
[0089] The methods described herein can include administering to
the subject one or more additional components. As discussed above,
the additional component may be provided in a separate composition
or may be combined with the .beta.-glucan composition, the second
composition, or both.
[0090] Suitable additional components can include anti-cancer drugs
such as, for example, doxorubicin, cisplatin, and/or irinotecan.
Other suitable anti-cancer drugs include, for example, taxanes,
nitrogen mustards, ethylenimine derivatives, alkyl sulfonates,
nitrosoureas, triazenes, folic acid analogs, pyrimidine analogs,
purine analogs, vinca alkaloids, antibiotics, enzymes, platinum
coordination complexes, substituted urea, methyl hydrazine
derivatives, adrenocortical suppressants, or antagonists. More
specifically, the chemotherapeutic agents can include at least one
steroid, at least one progestin, at least one estrogen, at least
one antiestrogen, at least one androgen, or any combination
thereof. Even more specifically, the chemotherapy agents can
include azaribine, bleomycin, bryostatin-1, busulfan, carmustine,
chlorambucil, CPT-11, cyclophosphamide, cytarabine, dacarbazine,
dactinomycin, daunorubicin, dexamethasone, diethylstilbestrol,
doxorubicin, ethinyl estradiol, etoposide, fluorouracil,
fluoxymesterone, gemcitabine, hydroxyprogesterone caproate,
hydroxyurea, L-asparaginase, leucovorin, lomustine,
mechlorethamine, medroprogesterone acetate, megestrol acetate,
melphalan, mercaptopurine, methotrexate, methotrexate, mithramycin,
mitomycin, mitotane, phenyl butyrate, prednisone, procarbazine,
semustine streptozocin, tamoxifen, taxanes, taxol, testosterone
propionate, thalidomide, thioguanine, thiotepa, uracil mustard,
vinblastine, vincristine, or any combination of thereof.
[0091] Suitable additional components include, for example mCRP
antagonists. Membrane-bound complement regulatory proteins (mCRPs)
can limit complement-dependent cytotoxicity. Some tumors are known
to overexpress one or more mCRPs, thereby interfering with
complement-dependent cytotoxicity directed against the tumor.
Membrane-bound complement regulatory proteins include, for example,
CD46, CD55 and CD59.
[0092] CD46 (membrane cofactor protein, MCP) inhibits C3b activity.
It serves as a receptor for seven human pathogens but was initially
discovered as a widely expressed C3b- and C4b-binding protein. It
was later shown to be a cofactor for the serine protease factor I
to inactivate by limited proteolysis C3b- and C4b-binding proteins
and components of the convertases. CD46 is involved in protecting
normal cells from excessive complement activation. A heterozygous
deficiency of CD46 can predispose an individual to hemolytic uremic
syndrome.
[0093] CD55 (decay accelerating factor, DAF) inhibits C3
convertases. It was first recognized as a species restricting
factor operating at the level of C3 activation. It binds C3b and
C4b to inhibit the formation and reduce the half-life of the C3
convertases. CD55 is broadly distributed among cells in contact
with serum, including both hematopoietic and non-hematopoietic
cells. Expression of CD55 on NK cells or their targets has been
associated with reduced efficiency of cell lysis. CD55 is involved
in regulating the deposition of C3 on nucleated cells.
[0094] CD59 (1F-5Ag, H19, HRF20, MACIF, MIRL, P-18, Protectin) can
inhibit the formation of the membrane attack complex (MAC). CD59
can block the formation of MAC by preventing development of a
hydrophobic region on the C9 molecule, important for insertion into
the target cell membrane. A single C9 molecule may bind to the
C5b-8 complex, but CD59 can interfere with unfolding of the C9
molecule that is necessary to permit binding of multiple subsequent
C9 molecules and MAC assembly. In addition, CD59 can prevent ion
channel formation by the C5b-8 complex and the small-size MAC
C5b-C9 complex, which in turn prevents leaky pore formation.
[0095] The methods described herein can be effective for
ameliorating at least one symptom or clinical sign associated with
cancer such as, for example, blood in the urine; pain and/or
burning upon urination; frequent urination; cloudy urine; other
problems with urination; pain in the bone and/or swelling around
the affected site; bone fractures; weakness; fatigue; repeated
infections; nausea; vomiting; constipation; bumps and/or bruises
that persist; dizziness; drowsiness; abnormal eye movements and/or
changes in vision; weakness and/or loss of feeling in the arms
and/or legs; difficulties walking; fits and/or convulsions; changes
in personality, memory and/or speech; headaches that tend to be
worse in the morning and ease during the day, accompanied by nausea
and/or vomiting; a lump or thickening of the breast; discharge from
the nipple; change in the skin of the breast; a feeling of heat
and/or enlarged lymph nodes under the arm; rectal bleeding (red
blood in stools or black stools); abdominal cramps; constipation
alternating with diarrhea; unexplained weight loss; loss of
appetite; pallid complexion; dull ache and/or pain in the back
and/or side; a lump in kidney area, sometimes accompanied by high
blood pressure and/or abnormality in red blood cell count; fever
and/or flu-like symptoms; bruising and/or prolonged bleeding;
enlarged lymph nodes, spleen, and/or liver; pain in bones and/or
joints; frequent infections; night sweats; wheezing; persistent
cough for months; blood-streaked sputum; persistent ache in chest;
congestion in lungs; change in mole and/or other bump on the skin,
including bleeding or change in size, shape, color, or texture;
painless swelling in the lymph nodes in the neck, underarm, or
groin; persistent fever; itchy skin and/or rash; small lumps in
skin; abdominal swelling; a lump in the mouth; ulceration of the
lip, tongue and/or inside of the mouth that does not heal within a
couple of weeks; dentures that no longer fit well; oral pain; foul
breath; loose teeth; changes in speech; abnormal vaginal bleeding;
digestive discomfort; intolerance of fatty foods; yellowing of the
skin; abdominal masses; tenderness over the bladder; indigestion or
heartburn; bloating after meals; a watery bloody discharge in
postmenopausal women; pain during sexual intercourse; and/or pelvic
pain.
[0096] Accordingly, the methods described herein can allow the
.beta.-glucan and the second composition to be administered in a
combination that improves efficacy of the active agent (i.e., EGF-r
antagonist or antibody that binds to an antigen specific to
KRAS-mutated cells) in the second composition and may reduce the
likelihood and/or extent of undesirable side effects of the active
agent in the second composition. Side effects associated with
administering an EGF-r antagonist include, for example, airway
obstruction, including bronchospasm, stridor, or hoarseness;
urticaria; hypotension; interstitial lung disease; inflammation;
renal pathology; acneform rash; dry skin; skin fissures, fever;
sepsis; kidney failure; pulmonary embolus; dehydration; diarrhea;
abdominal pain; vomiting; and/or inflammatory and/or infectious
sequelae such as, for example plepharitis, cheilitis, cellulites
and/or cyst. Side-effects associated with the administration of
EGF-r antagonist may be reduced in severity and/or frequency by
co-administering the EGF-r antagonist with .beta.-glucan.
[0097] When the methods described herein involve administering a
.beta.-glucan composition and a second composition to a subject,
the administration of the compositions can include sequential or
simultaneous administration. In embodiments in which the
compositions are combined prior to being administered to the
subject, simultaneous administration is self-evident. However,
simultaneous or substantially simultaneous--administration of the
compositions also can occur when the .beta.-glucan composition and
the second composition are separate. Substantially simultaneous
administration can be accomplished, for example, by administering
to the subject a single infusion that includes both a .beta.-glucan
and the second composition or in multiple, substantially
simultaneous injections in which each injection includes
administering a single composition. When administered separately,
each composition can independently be administered by the same
route or by different routes. For example, the .beta.-glucan
composition may be administered orally, while the second
composition can be administered intravenously.
[0098] In some embodiments, the .beta.-glucan can be administered
at a dosage of, generally, from about 0.01 mg/kg/day to about 10
mg/kg/day, although the methods described herein can be practiced
by administering the .beta.-glucan at a daily dosage outside of
this range. In some embodiments, the dose off .beta.-glucan can be
from about 0.1 mg/kg/day to about 2.5 mg/kg/day. In some
embodiments, the .beta.-glucan is PGG.
[0099] In some embodiments, the active agent of the second
composition can be administered at a dosage of, generally, from
about 100 mg/m.sup.2/week to about 800 mg/m.sup.2/week such as, for
example, from about 250 mg/m.sup.2/week to about 400
mg/m.sup.2/week. In one particular embodiment, cetuximab is
administered in a first infusion at a dose of from about 200
mg/m.sup.2 to about 400 mg/m.sup.2, followed by a weekly infusion
of from about 125 mg/m.sup.2 to about 150 mg/m.sup.2.
[0100] When the .beta.-glucan composition and the second
composition (e.g., an EGF-r antagonist composition) are
administered together, the .beta.-glucan composition and the second
composition may be administered in a single weekly infusion. Doses
of .beta.-glucan administered per week can range from about 0.07
mg/kg/week to about 200 mg/kg/week. Dosages of EGF-r antagonist can
vary. For example, cetuximab (ERBITUX, Bristol-Myers Squibb, New
York, N.Y.) is typically administered intravenously at a dose of
about 400-500 mg/m.sup.2 (about 10.8-13.5 mg/kg). As another
example, erlotinib (TARCEVA, Genentech, South San Francisco,
Calif.) is typically administered orally at a dose of about 100
mg/day (about 10 mg/kg/week for a 70 kg patient). The .beta.-glucan
and EGF-r antagonist may also be administered according to any
suitable schedule. Suitable dosing schedules include, for example,
one to three doses per week.
[0101] In one particular embodiment, the method includes therapy
for a proliferative disorder and includes administering PGG as the
.beta.-glucan and cetuximab as the second composition. In another
embodiment, the method includes administering PGG, cetuximab, and
irinotecan. In another embodiment, the method includes
administering PGG, cetuximab, and cisplatin. In another embodiment,
the method includes administering PGG, cetuximab, and
doxorubicin.
[0102] The .beta.-glucan composition and the second composition can
be administered separately or co-formulated in a suitable
co-formulated dosage form. A variety of preparations can be used to
formulate pharmaceutical compositions containing .beta.-glucan the
second composition. Techniques for formulation and administration
may be found in "Remington: The Science and Practice of Pharmacy,
Twentieth Edition," Lippincott Williams & Wilkins,
Philadelphia, Pa. Suitable formulations include, for example,
tablets, capsules, pills, powders, granules, dragees, gels,
slurries, ointments, solutions suppositories, injections,
inhalants, and aerosols. The formulations can be administered
locally or systemically. Also, the formulations may be administered
to provide for sustained release and/or depot of the active agent.
Suitable formulations can further include one or more
pharmaceutical excipients such as, for example, antioxidants,
stabilizing agents, wetting agents, clarifying agents,
viscosity-increasing agents, carriers, tonicity agents, buffers,
preservatives, and/or encapsulation formulations.
[0103] A carrier can be a solvent or dispersion medium containing,
for example, water, ethanol, polyol (for example, glycerol,
propylene glycol, and liquid polyethylene glycol, and the like),
vegetable oils, or mixtures or combinations thereof. The proper
fluidity can be maintained by, for example, the use of a coating
(e.g., lecithin), the maintenance of the required particle size in
the case of dispersion, and the use of surfactants. The carrier can
further include one or more antibacterial and antifungal agents
such as, for example, parabens, chlorobutanol, phenol, sorbic acid,
thimerosal, and mixtures or combinations thereof. In some cases, it
may be desirable to include isotonic agents such as, for example,
sugars or sodium chloride. Absorption of injectable compositions
can be sustained by including absorption delaying agents delaying
such as, for example, aluminum monostearate and gelatin in the
composition.
[0104] Suitable preservatives include, for example, benzalkonium
chloride, benzethonium chloride, chlorobutanol, thimerosal, and
mixtures and combinations thereof. Suitable buffers include, for
example, boric acid, sodium and potassium bicarbonate, sodium and
potassium borates, sodium and potassium carbonate, sodium acetate,
sodium biphosphate, and mixtures and combinations thereof. A buffer
(or mixture or combination of buffers) can be provided in amounts
sufficient to maintain the pH of the composition between about pH 6
and pH 8 such as, for example, between about pH 7 and pH 7.5.
Suitable tonicity agents include, for example, dextran 40, dextran
70, dextrose, glycerin, potassium chloride, propylene glycol,
sodium chloride, and mixtures or combinations thereof. A tonicity
agent may be provided in an ophthalmic solution composition in an
amount so the sodium chloride equivalent of the ophthalmic solution
is in the range 0.9.+-.0.2%. Suitable antioxidants and stabilizers
include, for example, sodium bisulfate, sodium metabisulfite,
sodium thiosulfite, thiourea, and mixtures or combinations thereof.
Suitable wetting and clarifying agents include, for example,
polysorbate 80, polysorbate 20, poloxamer 282, tyloxapol, and
mixtures or combinations thereof. Suitable viscosity-increasing
agents include, for example, dextran 40, dextran 70, gelatin,
glycerin, hydroxyethylcellulose, hydroxmethylpropylcellulose,
lanolin, methylcellulose, petrolatum, polyethylene glycol,
polyvinyl alcohol, polyvinylpyrrolidone, carboxymethylcellulose,
and mixtures or combinations thereof.
[0105] The compositions can be formulated by dissolving,
suspending, or emulsifying the active agent--i.e., the
.beta.-glucan, EGF-r antagonist, antibody, and/or other active
agents--in an appropriate aqueous or nonaqueous solvent. Suitable
nonaqueous solvents include, for example, vegetable or similar oils
(e.g., sesame oil, peanut oil, etc.), synthetic aliphatic acid
glycerides, esters of higher aliphatic acids, and/or propylene
glycol. Suitable aqueous solutions include, for example, Hank's
solution, Ringer's solution, and/or physiological saline
buffer.
[0106] In some embodiments, the compositions may be formulated for
subcutaneous or intramuscular injection. In such embodiments, the
solvent may be DMSO, which can result in rapid penetration and,
therefore, delivery of high concentrations of the compositions to a
small area.
[0107] The subject treated by the methods of the invention can be a
mammal such as, for example, a human, although they may also be
applied to non-human mammals such as, for example, apes, monkeys,
dogs, mice, etc. Thus, the methods described herein invention can
be practiced in a veterinarian context.
[0108] The present invention is illustrated by the following
examples. It is to be understood that the particular examples,
materials, amounts, and procedures are to be interpreted broadly in
accordance with the scope and spirit of the invention as set forth
herein.
EXAMPLES
Example 1
[0109] A culture of S. cerevisiae is started and expanded stepwise
through a shake flask culture into a 250 L scale production
fermenter. The S. cerevisiae are grown in a glucose-ammonium
sulfate medium enriched with vitamins (e.g., folic acid, inositol,
nicotinic acid, pantothenic acid (calcium and sodium salt),
pyridoxine HCl, and thymine HCl) and trace metals (e.g., from
compounds such as ferric chloride, hexahydrate; zinc chloride;
calcium chloride, dihydrate; molybdic acid; cupric sulfate,
pentahydrate; and boric acid). An antifoaming agent such as
Antifoam 204 may also be added at a concentration of about
0.02%.
[0110] The production culture is maintained under glucose
limitation in a fed batch mode. During seed fermentation, samples
are taken periodically to measure the optical density of the
culture before inoculating the production fermenter. During
production fermentation, samples are also taken periodically to
measure the optical density of the culture. At the end of
fermentation, samples are taken to measure the optical density, the
dry weight, and the microbial purity.
[0111] If desired, fermentation may be terminated by raising the pH
of the culture to at least 11.5 or by centrifuging the culture to
separate the cells from the growth medium. In addition, depending
on the size and form of purified .beta.-glucan that is desired,
steps to disrupt or fragment the yeast cells may be carried out.
Any known chemical, enzymatic or mechanical methods, or surfactants
include, for example, octylthioglucoside, Lubrol PX, Triton X-100,
sodium lauryl sulfate (SDS), Nonidet P-40, Tween 20, and the like.
Ionic (anionic, cationic, amphoteric) surfactants (e.g., alkyl
sulfonates, benzalkonium chlorides, and the like) and nonionic
surfactants (e.g., polyoxyethylene hydrogenated castor oils,
polyoxyethylene sorbitol fatty acid esters, polyoxyethylene
sorbitan fatty acid esters, polyoxyethylene glycerol fatty acid
esters, polyethylene glycol fatty acid esters, polyoxyethylene
alkyl phenyl ethers, and the like) may also be used. The
concentration of surfactant will vary and depend, in part, on which
surfactant is used.
[0112] Extractions are usually carried out at temperatures between
about 70.degree. C. and about 90.degree. C. Depending on the
temperature, the reagents used and their concentrations, the
duration of each extraction is between about 30 minutes and about 3
hours.
[0113] After each extraction, the solid phase containing the
.beta.-glucan is collected using centrifugation or continuous-flow
centrifugation and resuspended for the subsequent step. The
solubilized contaminants are removed in the liquid phase during the
centrifugations, while the .beta.-glucan remains in the insoluble
cell wall material.
[0114] Yeast cell material may be subject to one or more
extractions such as, for example, four extractions. In the first
extraction, harvested yeast cells are mixed with 1.0 M NaOH and
heated to 90.degree. C. for approximately 60 minutes. The second
extraction is an alkaline/surfactant extraction whereby the
insoluble material is resuspended in 0.1 M NaOH and about 0.5% to
0.6% Triton X-100 and heated to 90.degree. C. for approximately 120
minutes. The third extraction is an alkaline/surfactant extraction
whereby the insoluble material is resuspended in 0.1 M NaOH and
about 0.05% Triton X-100 and heated to 90.degree. C. for
approximately 60 minutes. In the fourth extraction, the insoluble
material is resuspended in about 0.5% Triton-X 100 and heated to
75.degree. C. for approximately 60 minutes.
[0115] The alkaline and/or surfactant extractions solubilize and
remove some of the extraneous yeast cell materials. The alkaline
solution hydrolyzes proteins, nucleic acids, mannans, and lipids.
Surfactant enhances the removal of lipids and other hydrophobic
impurities, which provides an additional advantage yielding an
improved .beta.-glucan product.
[0116] Fat content of the yeast S. cerevisiae produced by aerobic
and anaerobic growth ranges from about 3% to about 8%. The fat
content varies depending on growth conditions of the yeast. Table 1
provides an overview of the typical fat composition of the yeast S.
cerevisiae. The data are from the following references: [0117]
Blagovic, B., J. Rpcuc, M. Meraric, K. Georgia and V. Maric. 2001.
Lipid composition of brewer's yeast. Food Technol. Biotechnol.
39:175-181. [0118] Shulze. 1995. Anaerobic physiology of
Saccharomyces cerevisiae. Ph.D. Thesis, Technical University of
Denmark. [0119] Van Der Rest, M. E., A. H. Kamming, A Nakano, Y.
Anrak, B. Poolman and W. N. Koning. 1995. The plasma membrane of
Saccharomyces cerevisiae: structure, function and biogenesis.
Microbiol. Rev. 59:304-322.
TABLE-US-00001 [0119] TABLE 1 Shulze Blagovic et al (2001) Van Der
Rest Fatty acid (1995) (anaerobic growth) et al (1995) 10:0 Capric
acid 1.1% 12:0 and 12:1 4.8% 7.3% Lauric acid 14:0 and 14:1 8.8%
5.1% 7.0*% Myristic acid 16:0 Palmitic acid 26.8% 44.2% 12.8% 16:1
16.6% 16.9% 32.3% Palmitoleic acid 18:0 Stearic acid 6.1% 13.9%
8.0% 18.1 Oleic acid 25.7% 7.3% 28.0% 18:2 and higher 10.1% 5.3%
9.4% Linoleic acid, arachadonic acid and others *includes lipids
10:0 to 14:1
[0120] Yeast cell wall material typically contains 10-25% fat
depending on yeast type and growth conditions. Presently, during
processing of yeast cell wall material into .beta.-glucan, some,
but not all fat is removed by centrifugation and wash steps. Thus,
a typical preparation might yield a fat content of 3-7%.
[0121] The manufacturing process typically involves steps to remove
mannoproteins, lipids and other undesirable components of the yeast
cell wall. Some key steps common to this processing are various
wash steps that employ acid and alkali in separate washing steps to
liberate certain cell wall components. Several of the steps use an
alkali wash process where an alkali, usually sodium hydroxide, is
added to the liquid cell wall suspension. One of the purposes of
the alkali is to remove lipid by forming the free fatty acids of
the lipid. The result is a reduction in fat content of the
.beta.-glucan.
[0122] The alkali wash steps commonly used in production of yeast
.beta.-glucan leave behind residual fatty acids and partially
degraded fat triglycerides that have increased reactivity. The
direct result of the alkali wash process is the release of reactive
free fatty acids that quickly decompose to various oxidative
products of fat decomposition.
[0123] The next step in the purification process is an acidic
extraction that removes glycogen. One or more acidic extractions
are accomplished by adjusting the pH of the alkaline/surfactant
extracted material to between about pH 5 and pH 9 and mixing the
material in about 0.05 M to about 1.0 M acetic acid at a
temperature between about 70.degree. C. and 100.degree. C. for
approximately 30 minutes to about 12 hours.
[0124] The insoluble material remaining after centrifugation of the
alkaline/surfactant extraction is resuspended in water, and the pH
of the solution is adjusted to about 7 with concentrated HCl. The
material is mixed with enough glacial acetic acid to make a 0.1 M
acetic acid solution, which is heated to 90.degree. C. for
approximately 5 hours.
[0125] Next, the insoluble material is washed. In a typical wash
step, the material is mixed in purified water at about room
temperature for a minimum of about 20 minutes. The water wash is
carried out two times. The purified .beta.-glucan product is then
collected. Again, collection is typically carried out by
centrifugation or continuous-flow centrifugation.
[0126] At this point, a purified, particulate .beta.-glucan product
is formed. The product may be in the form of whole glucan particles
or any portion thereof, depending on the starting material. In
addition, larger sized particles may be broke down into smaller
particles. The range of product includes .beta.-glucan particles
that have substantially retained in vivo morphology (whole glucan
particles) down to submicron-size particles.
[0127] Particulate .beta.-glucan can be processed further to form
aqueous, soluble .beta.-glucan.
Example 2
[0128] Particulate .beta.-glucan such as, for example, particulate
.beta.-glucan as produced in EXAMPLE 1 may be further processed to
form soluble .beta.-glucan. The particulate .beta.-glucan starting
material may range in size from whole glucan particles down to
submicron-sized particles.
[0129] Particulate .beta.-glucan undergoes an acidic treatment
under pressure and elevated temperature to produce soluble
.beta.-glucan. Pelleted particulate .beta.-glucan is resuspended
and mixed in a sealable reaction vessel in a buffer solution and
brought to pH 3.6. Buffer reagents are added such that every liter,
total volume, of the final suspension mixture contains about 0.61 g
sodium acetate, 5.24 ml glacial acetic acid and 430 g pelleted
particulate .beta.-glucan. The vessel is purged with nitrogen to
remove oxygen and increase the pressure within the reaction
vessel.
[0130] The pressure inside the vessel is brought to 35 PSI, and the
suspension is heated to about 135.degree. C. for between about 4.5
and 5.5 hours. The pressure and temperature reduces the need to use
hazardous chemicals to solubilize the .beta.-glucan and thereby
improves safety and reduces costs for materials.
[0131] The exact duration of heat treatment is typically determined
experimentally by sampling reactor contents and performing gel
permeation chromatography (GPC) analyses. The objective is to
maximize the yield of soluble material that meets specifications
for high resolution-GPC(HR-GPC) profile and impurity levels, which
are discussed below. Once the .beta.-glucan is solubilized, the
mixture is cooled to stop the reaction.
[0132] The crude, solubilized .beta.-glucan may be washed and
utilized in some applications at this point, however, for
pharmaceutical applications further purification is performed. Any
combination of one or more of the following steps may be used to
purify the soluble .beta.-glucan. The soluble .beta.-glucan may be
clarified. Suitable clarification means include, for example,
centrifugation or continuous-flow centrifugation.
[0133] Next, the soluble .beta.-glucan may be filtered using, for
example, a depth filter followed by a 0.2 .mu.m filter.
[0134] The .beta.-glucan may be further purified by chromatography.
The soluble .beta.-glucan may be conditioned at some point during
previous steps in preparation for chromatography. For example, if a
chromatographic step includes hydrophobic interaction
chromatography (HIC), the soluble .beta.-glucan can be conditioned
to the appropriate conductivity and pH with a solution of ammonium
sulphate and sodium acetate. A suitable solution is 3.0 M ammonium
sulfate, 0.1 M sodium acetate, which is used to adjust the pH to
5.5.
[0135] In one example of HIC, a column is packed with Tosah
Toyopearl Butyl 650M resin (or equivalent). The column is packed
and qualified according to the manufacturer's recommendations.
[0136] Prior to loading, the column equilibration flow-through is
sampled for pH, conductivity and endotoxin analyses. The soluble
.beta.-glucan, conditioned in the higher concentration ammonium
sulphate solution, is loaded and then washed. The soluble
.beta.-glucan will bind to the HIC column. Low molecular weight
products as well as some high molecular weight products are washed
through. Soluble .beta.-glucan remaining on the column is eluted
with a buffer such as 0.2 M ammonium sulfate, 0.1 M sodium acetate,
pH 5.5. Multiple cycles may be necessary to ensure that the hexose
load does not exceed the capacity of the resin. Fractions are
collected and analyzed for the soluble .beta.-glucan product.
[0137] Another chromatographic step that may be utilized is gel
permeation chromatography (GPC). In one example of GPC, a Tosah
Toyopearl HW55F resin, or equivalent is utilized and packed and
qualified as recommended by the manufacturer. The column is
equilibrated and eluted using citrate-buffered saline (0.14 M
sodium chloride, 0.011 M sodium citrate, pH 6.3). Prior to loading,
column wash samples are taken for pH, conductivity and endotoxin
analyses. Again, multiple chromatography cycles may be needed to
ensure that the load does not exceed the capacity of the
column.
[0138] The eluate is collected in fractions, and various
combinations of samples from the fractions are analyzed to
determine the combination with the optimum profile. For example,
sample combinations may be analyzed by HR-GPC to yield the
combination having an optimized HR-GPC profile. In one optimized
profile, the amount of high molecular weight (HMW) impurity, that
is soluble .beta.-glucans over 380,000 Da, is less than or equal to
10%. The amount of low molecular weight (LMW) impurity, under
25,000 Da, is less than or equal to 17%. The selected combination
of fractions is subsequently pooled.
[0139] At this point, the soluble .beta.-glucan is purified and
ready for use. Further filtration may be performed in order to
sterilize the product. If desired, the hexose concentration of the
product can be adjusted to about 1.0.+-.0.15 mg/ml with sterile
citrate-buffered saline.
[0140] The soluble .beta.-glucan has an average molecular weight
between about 120,000 Da and about 205,000 Da and a molecular
weight distribution (polydispersity) of not more than 2.5 as
determined by HR-GPC with multiple angle light scattering
(HR-GPC/MALS) and differential refractive index detection. Powder
X-ray diffraction and magic-angle spinning NMR determined that the
product consists of polymeric chains associated into triple
helices. The soluble .beta.-glucan is typically uncharged and
therefore has no pK.sub.a. It is soluble in water independent of
pH, and the viscosity increases as the concentration increases.
Example 3
Mice and Tumor Models
[0141] BALB/c nude (Nu/Nu) mice were purchased from Taconic Farms,
Inc. (Hudson, N.Y.). For the GEO colon carcinoma xenograft model,
6- to 8-week-old SCID mice were implanted subcutaneously in a
mammary fatpad with 10.times.10.sup.6 GEO colon carcinoma cells,
which contain a Gly12Ala homozygous KRAS mutation. When a palpable
tumors reached 350 mm.sup.2, animals were divided into four groups
(n=7) and treated with either saline (control), ERBITUX
(Bristol-Myers Squibb, New York, N.Y., 0.25 mg/dose), IMPRIME PGG
(Biolpolymer Engineering, Inc., Eagan, Minn., 1.2 mg/dose), or
ERBITUX (0.25 mg/dose) and IMPRIME PGG (1.2 mg/dose), every three
days for eight doses.
[0142] During the treatment period, tumor volumes were calculated
as the average of perpendicular diameters measured using calipers.
Tumor volumes were determined every three days. Mice were
sacrificed when tumors reached 12 mm in diameter. Results are shown
in FIG. 1.
Example 4
Cell Culture
[0143] The tumor target line HCT-116 colorectal carcinoma (American
Type Culture Collection, Manassas, Va., ATCC No. CCL-247) was
maintained in McCoy's 5A media supplemented with 10% fetal calf
serum (Hyclone Laboratories, Inc., Logan, Utah).
Cytotoxicity Assay
[0144] Cytotoxicity was measured with the LIVE/DEAD Cell Mediated
Cytotoxicity Kit (Invitrogen, Carlsbad, Calif.) according to
manufacturer's instructions. Briefly, the HCT-116 target cells were
stained with 1:80 dilution of DiOC18 in Phosphate Buffered Saline
(PBS, Mediatech Inc. Manassas, Va.) and incubated at 37.degree. C.
for 30 minutes. The cells were then washed twice in PBS and
resuspended at 2.times.10.sup.6 cells/mL in complete media
containing 10% human serum.
[0145] DiOC labeled target cells incubated with ERBITUX dilutions
for 30 minutes at 37.degree. C. In each well of a 96-well plate,
1.25.times.10.sup.6 freshly isolated peripheral blood mononuclear
cells (PBMC) along with IMPRIME-PGG dilution or 50 ng/ml IL-2
(R&D Systems, Minneapolis, Minn.) in of total volume of 100
.mu.l media incubated at 37.degree. C. for 30 minutes.
5.times.10.sup.4 targets added to wells in 25 .mu.l of media and
assay incubated overnight at 37.degree. C. Following incubation,
target cell viability measured by staining with 125 .mu.l of
propidium iodide (PI) viability reagent diluted 1:5000 from stock
solution provided in the assay kit. Cells incubated for 30 minutes
at 37.degree. C. before analysis on LSR II flow cytometer (BD
Biosciences, San Jose, Calif.) to determine percentage of DiOC
stained targets that intercalate the PI stain, indicating cell
death. Results are shown in FIGS. 2-4.
[0146] The complete disclosure of all patents, patent applications,
and publications cited herein are incorporated by reference in
their entirety. In the event that any inconsistency exists between
the disclosure of the present application and the disclosure of any
document incorporated herein by reference, the disclosure of the
present application shall govern. The foregoing detailed
description and examples have been given for clarity of
understanding only. No unnecessary limitations are to be understood
therefrom. The invention is not limited to the exact details shown
and described, for variations obvious to one skilled in the art
will be included within the invention defined by the claims.
[0147] Unless otherwise indicated, all numbers expressing
quantities of components, molecular weights, and so forth used in
the specification and claims are to be understood as being modified
in all instances by the term "about." Accordingly, unless otherwise
indicated to the contrary, the numerical parameters set forth in
the specification and claims are approximations that may vary
depending upon the desired properties sought to be obtained by the
present invention. At the very least, and not as an attempt to
limit the doctrine of equivalents to the scope of the claims, each
numerical parameter should at least be construed in light of the
number of reported significant digits and by applying ordinary
rounding techniques.
[0148] Notwithstanding that the numerical ranges and parameters
setting forth the broad scope of the invention are approximations,
the numerical values set forth in the specific examples are
reported as precisely as possible. All numerical values, however,
inherently contain a range necessarily resulting from the standard
deviation found in their respective testing measurements.
[0149] All headings are for the convenience of the reader and
should not be used to limit the meaning of the text that follows
the heading, unless so specified.
* * * * *